The present invention relates to a novel interleukin-1 receptor signaling protein. More specifically, isolated nucleic acid molecules are provided encoding a human interleukin-1 receptor associated kinase-2 (IRAK-2). IRAK-2 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same.
Interleukin-1 (IL-1).
Interleukin-1 (IL-1xcex1 and IL-1xcex2) is a xe2x80x9cmultifunctionalxe2x80x9d cytokine that affects nearly every cell type, and often in concert with other cytokines or small mediator molecules. (Dinarello, C. A., Blood 87:2095-2147 (Mar. 15, 1996).) There are three members of the IL-1 gene family: IL-1xcex1, IL-1xcex2, and IL-1 receptor antagonist (IL-1Ra). IL-1xcex1 and IL-1xcex2 are agonists and IL-1Ra is a specific receptor antagonist. IL-1xcex1 and xcex2 are synthesized as precursors without leader sequences. The molecular weight of each precursor is 31 kD. Processing of IL-1xcex1 or IL-1xcex2 to xe2x80x9cmaturexe2x80x9d forms of 17 kD requires specific cellular proteases. In contrast, IL-1Ra evolved with a signal peptide and is readily transported out of the cells and termed secreted IL-1Ra (sIL-1Ra).
IL-1 Receptor and Ligands.
The receptors and ligands of the IL-1 pathway have been well defined (for review, see Dinarello, C. A., FASEB J. 8:1314-1325 (1994); Sims, J. E. et al., Interleukin-1 signal transduction: Advances in Cell and Molecular Biology of Membranes and Organelles, Vol. 3, JAI Press, Inc., Greenwich, Conn. (1994), pp. 197-222). Three ligands, IL-1xcex1, IL-1xcex2, and IL-1 receptor antagonist (IL-1Ra) bind three forms of IL-1 receptor, an 80-kDa type I IL-1 receptor (IL-1RI) (Sims, J. E. et al., Science 241:585-589 (1988)), a 68-kDa type II IL-1 receptor (IL-1RII) (McMahan, C. J. et al., EMBO J. 10:2821-2832 (1991)), and a soluble form of the type II IL-1R (sIL-1RII) (Colotta, F. et al., Science 261:472-475 (1993)).
IL-1 Production in Various Disease States.
Increased IL-1 production has been reported in patients with various viral, bacterial, fungal, and parasitic infections; intravascular coagulation; high-dose IL-2 therapy; solid tumors; leukemias; Alzheimer""s disease; HIV-1 infection; autoimmune disorders; trauma (surgery); hemodialysis; ischemic diseases (myocardial infarction); noninfectious hepatitis; asthma; UV radiation; closed head injury; pancreatitis; periodontitis; graft-versus-host disease; transplant rejection; and in healthy subjects after strenuous exercise. There is an association of increased IL-1xcex2 production in patients with Alzheimer""s disease and a possible role for IL-1 in the release of the amyloid precursor protein (Vasilakos, J. P., et al., FEBS Lett. 354:289 (1994)). However, in most conditions, IL-1 is not the only cytokine exhibiting increased production and hence the specificity of the IL-1 findings as related to the pathogenesis of any particular disease is lacking. In various disease states, IL-1xcex2, but not IL-1xcex1, is detected in the circulation.
IL-1 in Therapy.
Although IL-1 has been found to exhibit many important biological activities, it is also found to be toxic at doses that are close to therapeutic dosages (Dinarello, C. A., Blood 87:2095-2147 (Mar. 15, 1996)). In general, the acute toxicities of either isoform of IL-1 were greater after intravenous compared with subcutaneous injection. Subcutaneous injection was associated with significant local pain, erythema, and swelling (Kitamura, T., and Takaku, F., Exp. Med. 7:170 (1989); Laughlin, M. J., Ann. Hematol. 67:267 (1993)). Patients receiving intravenous IL-1 at doses of 100 ng/kg or greater experienced significant hypotension. In patients receiving IL-1xcex2 from 4 to 32 ng/kg subcutaneously, there was only one episode of hypotension at the highest dose level (Laughlin, M. J., Ann. Hematol. 67:267 (1993)).
Contrary to IL-1-associated myelostimulation in patients with normal marrow reserves, patients with aplastic anemia treated with 5 daily doses of IL-1xcex1 (30 to 100 ng/kg) had no increases in peripheral blood counts or bone marrow cellularity (Walsh, C. E., et al., Br. J. Haematol 80:106 (1992)). IL-1 has been administered to patients undergoing various regiments of chemotherapy to reduce the nadir of neutropenia and thrombocytopenia.
Daily treatment with 40 ng/kg IL-1xcex1 from day 0 to day 13 of autologous bone marrow or stem cells resulted in an earlier recovery of neutropenia (median, 12 days; P less than 0.001) (Weisdorf, D., et al., Blood 84:2044 (1994)). After 14 days of treatment, the bone marrow was significantly enriched with committed myeloid progenitor cells. Similar results were reported in patients with AML receiving 50 ng/kg/d of IL-1xcex2 for 5 days starting at the time of transplantation with purged or nonpurged bone marrow (Nemunaitis, J., et al., Blood 83:3473 (1994)). Injecting humans with low doses of either IL-1xcex1 or IL-1xcex2 confirms the impressive pyrogenic and hypotension-inducing properties of the molecules.
IL-1 Signaling Mechanisms.
After binding to interleukin-1 (IL-1), the IL-1 receptor type I (IL-1RI) associates with the IL-1R Accessory Protein (IL-1RAcP) and initiates a signaling cascade that results in the activation of NF-kB, (Greenfeder, S. A., et al., J. Biol. Chem. 270:13757-65 (1995); Sims, J. E., et al., Science 241:585-9 (1988); Korherr, C., et al., Eur. J. Immunol. 27:262-7 (1997); Wesche, H., et al., J. Biol. Chem. 272:7727-31 (1997); Freshney, N. W., et al., Cell 78:1039-49(1994); and Martin, M., et al., Eur. J Immunol. 24:1566(1994)). Significant similarity exists between the IL-1R signaling pathway in mammals and the Toll signaling pathway in Drosophila. Toll, which shares sequence homology with the cytoplasmic domain of the IL-1RAcP, induces Dorsal activation (a homologue of NF-kB) via the adapter protein Tube and the protein kinase Pelle, (Galindo, R. L., et al., Development 121:2209-18 (1995); Norris, J. L. and Manley, J. L., Genes Devel. 10:862-72 (1996); Letsou, A., et al., EMBO 12:3449-3458 (1993); and Grosshans, J., et al., Nature 372:563-566 (1994)); significantly the recently identified IRAK (IL-1R Associated Kinase) is homologous to Pelle, (Cao, Z., et al., Science 271:1128-31 (1996)). However, in mammalian cells, additional complexity is thought to exist based on the observation that multiple protein kinase activities coprecipitate with the IL-1RI (Singh, R., et al., J. Clin. Invest. 100:419 (1997); and Eriksson, A., et al., Cytokine 7:649 (1995)). Furthermore, given that in Drosophila the adapter protein Tube interacts with and regulates Pelle""s activity, it is likely that analogous adapter/regulatory molecules might participate in IL-1 signaling. There is a need in the art to characterize molecules involved in the IL-1 signaling pathway.
Nuclear Factor Kappa B (NF-kB).
NF-kB is a member of a family of dimeric transcription factors made from monomers that have approximately 300 amino-acid Rel regions which bind to DNA, interact with each other, and bind the IkB inhibitors (for review, see Baeuerle and Baltimore, Cell 87:13-20 (1996)). Disregulation of NF-kB has been implicated in malignant transformation and hyperplasia (Gilmore et al., Oncogene 9:2391-2398 (1996)). NF-kB plays an important role in the antiviral response as a virus-inducible transcriptional regulator of xcex2-interferon, MHC class I, and inflammatory cytokine genes. NF-kB has also been shown to protect cells from pro-apoptotic stimuli (Beg et al., Nature 376:167-170 (1995)).
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the IRAK-2 polypeptide having the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 209340 on Oct. 7, 1997.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of IRAK-2 polypeptides or peptides by recombinant techniques.
The invention further provides an isolated IRAK-2 polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by the IRAK-2, which involves contacting cells which express the IRAK-2 with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
The invention provides a diagnostic method useful during diagnosis of a IRAK-2 or IL-1 disorder.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of IRAK-2 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated IRAK-2 polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of IRAK-2 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of an IRAK-2 antagonist.